Bacillus thuringiensis strains active against lepidopteran and coleopteran pests

ABSTRACT

The invention is related to a novel biologically pure Bacillus thuringiensis (B.t.) strains active against lepidopteran and coleopteran pests which produces a bipyramidal crystal consisting essentially of at least two delta-endotoxins having a molecular weight of about 130,000 daltons and a rhomboidal crystal consisting essentially of two delta-endotoxins, each having a molecular weight of about 33,000 daltons, as well as spores, crystals, delta-endotoxins and/or mutants thereof. The invention also relates to insecticidal compositions obtainable therefrom. The invention further relates to methods of using the insecticidal compositions to control an insect pest(s) from the order Lepidoptera and/or Coleoptera. The invention also relates to isolated DNA sequences encoding the delta-endotoxins.

This application is a continuation-in-part of application Ser. No.08/264,100, filed Jun. 22, 1994, now abandoned, which is acontinuation-in-part of Ser. No. 08/194,651, filed Feb. 9, 1994, nowabandoned, which is a continuation-in-part of application Ser. No.08/166,391, filed Dec. 13, 1993, now abandoned which is acontinuation-in-part of application Ser. No. 07/991,073, filed Dec. 15,1992, now abandoned.

1. FIELD OF THE INVENTION

The invention is related to a novel biologically pure Bacillusthuringiensis (B.t.) strain(s) active against lepidopteran andcoleopteran pests which produces a bipyramidal crystal consistingessentially of at least two delta-endotoxins having a molecular weightof about 130,000 daltons and a rhomboidal crystal consisting essentiallyof two delta-endotoxins, each having a molecular weight of about 33,000daltons, as well as spores, crystals, delta-endotoxins and/or mutantsthereof. The invention also relates to insecticidal compositionsobtainable therefrom. The invention further relates to methods of usingthe insecticidal compositions to control an insect pest(s) from theorder Lepidoptera and/or Coleoptera. The invention also relates toisolated DNA sequences encoding the delta-endotoxins.

2. BACKGROUND OF THE INVENTION

Every year, significant portions of the world's commercially importantagricultural crops, including foods, textiles, and various domesticplants are lost to pest infestation, resulting in losses in the millionsof dollars. Various strategies have been used in attempting to controlsuch pests.

One strategy is the use of broad spectrum pesticides, chemicalpesticides with a broad range of activity. However, there are a numberof disadvantages to using such chemical pesticides. Specifically,because of their broad spectrum of activity, these pesticides maydestroy non-target organisms such as beneficial insects and parasites ofdestructive pests. Additionally, these chemical pesticides arefrequently toxic to animals and humans, and targeted pests frequentlydevelop resistance when repeatedly exposed to such substances.

Another strategy has involved the use of biopesticides, which make useof naturally occurring pathogens to control insect, fungal and weedinfestations of crops. Biopesticides are naturally occuring organismsthat produce a toxin(s), a substance toxic to the infesting agent whichis generally less harmful to non-target organisms and the environment asa whole than chemical pesticides.

The most widely used biopesticide is Bacillus thuringiensis (B.t.). B.t.is a widely distributed, rod shaped, aerobic and spore formingmicroorganism. During its sporulation cycle, B.t. produces a protein(s)known as a delta-endotoxin(s), that forms crystalline inclusion bodieswithin the cell. The delta-endotoxins have molecular weights rangingfrom 27-140 kD and kill insect larvae upon ingestion.

Delta-endotoxins have been produced by recombinant DNA methods (see, forexample, Tailor et al., 1992, Molecular Microbiology 6: 1211-1217; toxinis active against lepidopteran and coleopteran pests; Payne et al., U.S.Pat. No. 5,045,469; toxin is active against lepidopteran pests). Thedelta-endotoxins produced by recombinant DNA methods may or may not bein crystal form.

A number of B.t. strains have been isolated that have been found to beactive against insect pests of the order Lepidoptera. B.t. subsp.kurstaki HD-1 produces bipyramidal and cuboidal crystal proteins in eachcell during sporulation (Luthy et al., in Microbial and ViralPesticides, ed. E. Kurstak, Marcel Dekker, New York, 1982, pp. 35-74);the bipyramidal crystal was found to be encoded by three cryIA genes(Aronson et al., 1986, Microbiol. Rev. 50: 1-50). B.t. subsp. kurstakiHD-73 crystal delta-endotoxin contains the CryIA(c) protein (Adang etal., 1985, Gene 36: 289-300). B.t. subsp. dendrolimus HD-7 and HD-37contain a CryIA and a CryII protein; B.t. subsp. sotto contains analkaline soluble protein that differs from the holotype CryIA(a) proteinby 24 amino acids; B.t. subsp. subtoxicus HD-10 contains CryIA and CryIBproteins; B.t. subsp. tolworthi HD-121 contains CryIA and CryIIproteins; and B.t. subsp. aizawai HD-68 contains CryIA proteins (Hofteand Whiteley, 1989, Microbiol. Reviews 53: 242-255). Payne, U.S. Pat.No. 4,990,332, issued Feb. 5, 1993, discloses an isolate of B.t.,PS85AI, and a mutant of the isolate, PS85AI, which both have activityagainst Plutella xylostella, a lepidopteran pest, and produce alkalinesoluble proteins having a molecular weight of 130,000 and 60,000daltons. Payne, U.S. Pat. No. 5,045,469, issued Sep. 3, 1991 discloses aB.t. isolate designated PS81F which also produces alkaline solubleproteins having a molecular weight of 130,000 and 60,000 daltons and hasactivity against Spodoptera exigua and T. ni; the toxin gene from PS81Fappears to have little homology to the toxin gene from B.t. subsp.kurstaki HD-1. Payne, U.S. Pat. No. 5,206,166, filed Jun. 25, 1992,issued Apr. 27, 1993, discloses B.t. isolates PS81A2 and PS81RR1 whichproduce 133,601 and 133,367 dalton alkaline-soluble proteins; both haveactivity against Trichoplusia ni, Spodoptera exigua and Plutellaxylostella and are different from B.t. subsp. kurstaki HD-1 and otherB.t. isolates. Bernier et al., U.S. Pat. No. 5,061,489 and WO 90/03434discloses strain A20 producing a delta-endotoxin encoded by at leastthree genes: 6.6-, 5.3-, and 4.5-type genes (cryIA(a), cryIA(b), andcryIA(c)). Chestukhina et al., 1988, FEBS Lett. 232: 249-51, disclosethat B.t. subsp. galleriae produces two delta-endotoxins, both of whichare active against lepidopteran pests.

Other strains, e.g. Bacillus thuringiensis subsp. tenebrionis (Krieg etal., 1988, U.S. Pat. No. 4,766,203), have been found to be specific forColeoptera. The isolation of another coleopteran toxic Bacillusthuringiensis strain was reported in 1986 (Hernnstadt et al.Bio/Technology vol. 4, 305-308, 1986, U.S. Pat. No. 4,764,372, 1988).This strain, designated "Bacillus thuringiensis subsp. san diego", M-7,has been deposited at the Northern Regional Research Laboratory, USAunder accession number NRRL B-15939. However, the assignee of the '372patent, Mycogen, Corp. has publicly acknowledged that Bacillusthuringiensis subsp. san diego is Bacillus thuringiensis subsp.tenebrionis.

Other isolated strains have been found to be active against two ordersof pests. Padua, 1990, Microbiol. Lett. 66: 257-262, discloses theisolation of two mutants containing two delta-endotoxins, a 144 kDprotein having activity against a lepidopteran pest and a 66 kD proteinhaving activity against mosquitoes. Bradfish et al., U.S. Pat. No.5,208,017, discloses B.t. isolates PS86A1 and PS86Q3 which producealkaline soluble proteins having a molecular weight of 58,000 and 45,000daltons and 155,000, 135,000, 98,000, 62,000, and 58,000 daltons,respectively and which have activity against lepidopteran andcoleopteran pests. PCT Application No. WO 90/13651 and Tailor et al.,1992, Molecular Microbiology 6: 1211-1217, disclose a B.t. strain whichis toxic against Lepidoptera and Coleoptera and which produces a toxinhaving a molecular weight of 81 kd.

It is advantageous to isolate new strains of Bacillus thuringiensis toproduce new toxins so that there exists a wider spectrum ofbiopesticides for any given insect pest.

3. SUMMARY OF THE INVENTION

The invention is related to a novel biologically pure Bacillusthuringiensis strain(s) or a spore(s), crystal(s) or mutant(s) thereofwhich strain or mutant in contrast to B.t. strains disclosed in theprior art, has activity against an insect pest of the order Lepidopteraand an insect pest of the order Coleoptera, produces at least twodelta-endotoxins having a molecular weight of about 130,000 daltons andtwo delta-endotoxins both having molecular weights of about 33,000daltons. One of the 33,000 dalton delta-endotoxins has an amino acidsequence essentially as depicted in SEQ ID NO:37 (hereinafter referredto as the "MIVDL protein"). The other 33,000 dalton delta-endotoxin hasan amino acid sequence essentially as depicted in SEQ ID NO:38(hereinafter referred to as the "MKHHK protein"). The 130,000delta-endotoxins have insecticidal activity against insect pests of theorder Lepidoptera.

The invention also relates to each of the delta-endotoxins as well as anisolated nucleic acid fragment containing a nucleic acid sequenceencoding each of the delta-endotoxins or a portion of thedelta-endotoxin having insecticidal activity against a pest. In oneembodiment, the nucleic acid fragment contains a nucleic acid sequenceencoding the MIVDL protein and may have the nucleic acid sequenceessentially as depicted in SEQ ID NO:39. In another embodiment, thenucleic acid fragment contains a nucleic acid sequence encoding theMKHHK protein and may have the nucleic acid sequence essentially asdepicted in SEQ ID NO:40. The invention is also directed to a genomicsequence comprising nucleic acid sequence encoding the MKHHK and/orMIVDL and may have the nucleic acid sequence essentially as depicted inSEQ ID NOS:41 (MKHHK and MIVDL), 44 (MKHHK), and 45 (MIVDL).

The invention also provides vectors, DNA constructs and recombinant hostcells comprising the claimed nucleic acid fragment(s), which vectors,DNA constructs and recombinant host cells are useful in the recombinantproduction of the delta-endotoxins of the present invention. The nucleicacid fragment may be operably linked to transcription and translationsignals capable of directing expression of the delta-endotoxin in thehost cell of choice. Recombinant production of the delta-endotoxin(s) ofthe invention is achieved by culturing a host cell transformed ortransfected with the nucleic acid fragment of the invention, or progenythereof, under conditions suitable for expression of thedelta-endotoxin, and recovering the delta-endotoxin from the culture.

The invention is further related to an oligonucleotide probe having anucleotide sequence essentially as depicted in SEQ ID NO:20 which can beused to detect the MIVDL protein and and oligonucleotide probeessentially as depicted in SEQ ID NO:21 which can be used to detect theMKHHK protein.

In a specific embodiment of the invention, the Bacillus thuringiensisstrain of the present invention is EMCC0075 and EMCC0076 having theidentifying characteristics of NRRL B-21019 and NRRL B-21020respectively.

The novel Bacillus thuringiensis strains, spores, mutants or crystalsand/or delta-endotoxins may within the scope of this invention each beformulated into insecticidal compositions. In one embodiment, thestrain, spores, mutants, crystals, and/or delta-endotoxins may becombined with an insecticidal carrier. Insecticidal compositionscomprising the strains or mutants of the invention and/or spores, and/orcrystals thereof may be used to control insect pests of the orderLepidoptera and/or insect pests of the order Coleoptera in a methodcomprising exposing the pest to an insect-controlling effective amountof such an insecticidal composition.

Furthermore, the compositions or delta-endotoxins of the presentinvention may be used to enhance the insecticidal activity of anotherBacillus-related insecticide. As defined herein, "a Bacillus relatedinsecticide" is a Bacillus (e.g., Bacillus thuringiensis, specifically,Bacillus thuringiensis subsp. kurstaki or Bacillus thuringiensis subsp.tenebrionis or Bacillus subtilis) strain, spore, or substance, e.g.,protein or fragment thereof having activity against or which killinsects; a substance that provides plant protection, e.g. antifeedingsubstance; or a microorganism capable of expressing a Bacillus geneencoding a Bacillus protein or fragment thereof having activity againstor which kills insects (e.g., Bacillus thuringiensis delta-endotoxin)and an acceptable carrier (see Section 5.2., infra, for examples of suchcarriers). A microorganism capable of expressing a Bacillus geneencoding a Bacillus protein or fragment thereof having activity againstor which kill insects inhabits the phylloplane (the surface of the plantleaves), and/or the rhizosphere (the soil surrounding plant roots),and/or aquatic environments, and is capable of successfully competing inthe particular environment (crop and other insect habitats) with thewild-type microorganisms and provide for the stable maintenance andexpression of a Bacillus gene encoding a Bacillus protein or fragmentthereof having activity against or which kill insects. Examples of suchmicroorganisms include but are not limited to bacteria, e.g., generaBacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. familiesCyanophyceae, Prochlorophyceae, Rhodophyceae, Dinophyceae,Chrysophyceae, Prymnesiophyceae, Xanthophyceae, Raphidophyceae,Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae,Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g.,genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces,Rhodotorula, and Aureobasidium.

In a specific embodiment, the delta-endotoxins or compositions of thepresent invention may act together with Bacillus-related insecticides ina synergistic fashion. In another embodiment, Bacillus strains activeagainst insect pests of the order Coleoptera may act together in asynergistic fashion with delta-endotoxins, Bacillus strains or sporesthereof active against insect pests of the order Lepidoptera to killinsect pests of the order Coleoptera. In yet another embodiment, thedelta-endotoxins of the present invention may act together in asynergistic fashion.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of PCR analysis of Bacillus thuringiensisstrains for cryI genes by agarose gel electrophoresis. Lane 1 showsmolecular weight markers (1 kb ladder, BRL-GIBCO). Lanes 2 and 3 showanalysis of strains EMCC0075 and EMCC0076 with cryID oligonucleotideprimers described in FIG. 1. Lanes 4-6 show the analysis of Bacillusthuringiensis subsp. tenebrionis, an unknown Bacillus thuringiensisstrain, and Bacillus thuringiensis subsp. aizawai with cryIDoligonucleotide primers. Bacillus thuringiensis subsp. tenebrioniscontains only the cryIIIA gene; the unknown Bacillus thuringiensisstrain does not contain the cryID gene; and Bacillus thuringiensissubsp. aizawai contains several cryI genes including cryID.

FIG. 2 shows the cloned DNA fragments which encode the MKHHK and MIVDLproteins.

FIGS. 3A and 3B shows the homology of the "MIVDL" protein to the 34 kDaprotein of Bacillus thuringiensis subsp. thompsoni and the CryIA(a)protein of Bacillus thuringiensis subsp. kurstaki.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Obtaining Delta-Endotoxins

The spores and crystals of the present invention are obtainable from thestrains of the present invention. The strains of the present inventionmay be cultured using media and fermentation techniques known in the art(see, for example, Rogoff et al., 1969, J. Invertebrate Path. 14:122-129; Dulmage et al., 1971, J. nvertebrate Path. 18: 353-358; Dulmageet al., in Microbial Control of Pests and Plant Diseases, H. D. Burges,ed., Academic Press, N.Y., 1980). Upon completion of the fermentationcycle, the crystals and spores can be harvested by separating B.t.spores and crystals from the fermentation broth by means well known inthe art, e.g. centrifugation. The spores and crystals are contained inthe pellet.

As noted in Section 2, supra, crystals consist essentially of adelta-endotoxin(s). The strains of the present invention produce twotypes of crystals. One is a bipyramidal crystal consisting essentiallyof at least two 130,000 dalton delta-endotoxins. The other is arhomboidal crystal consisting essentially of the two 33,000 daltondelta-endotoxins.

Purification of the crystals or delta-endotoxins can be carried out byvarious procedures known in the art, including, but not limited to,density gradient centrifugation, chromatography (e.g. ion exchange,affinity, hydrophobic and size exclusion), electrophoretic procedures,differential solubility, or any other standard technique for thepurification of proteins.

The delta-endotoxins may also be obtained from a recombinant DNAexpression system. Specifically, DNA encoding each toxin as, forexample, essentially depicted in SEQ ID NOS:39, 40, 44, and 45 is clonedinto a suitable DNA expression vector. Alternatively one genomic DNAfragment comprising nucleic acid sequences encoding each delta endotoxinas, for example, essentially depicted in SEQ ID NO:41 may be cloned.

Identification of the specific DNA fragment encoding the delta-endotoxinmay be accomplished in a number of ways, including, but not limited to,electrophoretic separation of the fragments (Southern, 1975, J. Mol.Biol. 98: 503) in agarose, transfer of the separated DNA fragments tonitrocellulose, nylon, or other suitable support medium, and probing ofthe transferred fragments with a degenerate oligonucleotide probe(s)based on the amino acid sequence of the protein as determined bysequential Edman degradation. Alternatively, one may probe with alabeled gene fragment corresponding to the open reading frame of aprotein with suspected high homology to the protein of interest. Highhomology to the gene of interest may be determined by alignment of afamily of related proteins and identification of highly conservedregions in the encoding DNA segments (see, for example, Gribskov, K.,and J. Devereux, eds., in Sequence Analysis Primer, Stockton Press,N.Y., 1991). An elegant and reliable method is to determine the aminoacid sequences of at least two peptide fragments, generated by enzymaticor chemical means from the protein of interest, design degenerateoligonucleotides that will recognize the DNA encoding those regions, andthen to apply polymerase chain reaction (PCR) techniques to amplifyperfect or near-perfect copies of the intervening region of DNA. ThisPCR-generated segment of DNA can then be labeled and used as a highlyspecific probe for cloning the delta-endotoxin-encoding gene.

Once identified, the DNA fragment harboring the gene encoding thedelta-endotoxin or a portion thereof may be cloned by ligation of asize-selected library of fragments expected to harbor the gene ofinterest into a suitable vector, including, but not limited to, pBR322,pUC118, pACYC194, and PBCSK plasmids and their variants fortransformation into Escherichia coli; or pUB110, pBD64, pBC16, pHP13,pE194, pC194, and their variants, for transformation into Bacillus spp.Bacteriophage vectors, such as lambda and its derivatives, may also beused for cloning of the gene(s) into E. coli.

Production of the delta-endotoxin or a portion thereof at commerciallyuseful levels can be achieved by subcloning the encoding gene intoplasmid vectors that permit stable expression and maintenance in asuitable host. Frequently, acceptable expression can be achieved usingthe native regulatory elements present on the DNA fragment encoding thedelta-endotoxin. However, one might wish to add or alter transcriptionalregulatory signals (promoters, initiation start sites, operators,activator regions, terminators) and translational regulatory signals(ribosomal binding sites, initiation codons) for enhanced or moreregulated expression of the delta-endotoxin gene within the chosen hostcell.

In addition to plasmids, delta-endotoxin genes and the appropriateregulatory elements may be introduced into one of the native plasmids ofBacillus thuringiensis and/or other chosen host, or into the chromosomalDNA, via "gene conversion" (e.g., Iglesias and Trautner, 1983, Mol Gen.Genet. 189: 73-76; Duncan et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75: 3664-3665) or homologous recombination (e.g., Ferrari et al., 1983,J. Bacteriol. 154: 1513-1515) at sites of shared DNA homology betweenthe vector and the host strain. An efficient "two-plasmid" system may beused for introduction of genes into Bacilli via homologous recombination(see, for example, PCT Patent WO91/09129). Transposons may also be usedto introduce cry genes into the selected host strain. For example, inthe Bacilli, transposons such as Tn917 and its derivatives may be used(Youngman et al., 1989, In Regulation of Prokaryotic Development, I.Smith, R. Slepecky, and P. Setlow, eds. American Society forMicrobiology, Washington, D.C.).

Transfer of cloned delta-endotoxin genes into Bacillus thuringiensis, aswell as into other organisms, may be achieved by a variety oftechniques, including, but not limited to, protoplasting of cells (Changand Cohen, 1979, Mol. Gen. Genet. 168: 111-115; Crawford et al., 1987,J. Bacteriol. 169: 5423-5428); electroporation (e.g., Schurter et al.,1989, Mol. Gen. Genet. 218: 177-181 and Macaluso et al., 1991, J.Bacteriol. 173: 1353-1356); particle bombardment (e.g., Shark et al.,1991, Appl. Environ. Microbiol. 57: 480-485); silicon carbidefiber-mediated transformation of cells (Kaeppler et al., 1992, Theor.Appl. Genet. 84: 560-566); conjugation (Gonzalez et al., 1982, Proc.Natl. Acad. Sci. U.S.A. 79: 6951-6955); or transduction by bacteriophage(e.g., Lecadet et al., 1992, Appl. Environ. Microbiol. 58: 840-849).Transformed colonies may be detected by their ability to produce crystaldelta-endotoxin, to bind antibody directed against that specificdelta-endotoxin, or to kill susceptible pests, e.g., arthropods ornematodes, in bioassay.

Criteria for selection of a particular host for production include, butare not limited to, ease of introducing the gene into the host,availability of expression systems, and stable maintenance andexpression of the gene encoding the delta-endotoxin. The host may be amicroorganism, such as Bacillus thuringiensis itself, or an inhabitantof the phytosphere, e.g., the phylloplane (the surface of plants),and/or the rhizosphere (the soil surrounding plant roots), and/oraquatic environments, and should be capable of competing in theparticular environment (crop and other insect habitats) with thewild-type microorganisms. Examples of such microorganisms include butare not limited to bacteria, e.g. genera Bacillus, Pseudomonas, Erwinia,Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Met hylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes, andClostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae,Rhodophyceae, Dinophyceae, Chrysophyceae, Prymnesiophyceae,Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae,Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; andfungi, particularly yeast, e.g. genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.

The gene(s) encoding the delta-endotoxin(s) of the present invention ora portion thereof can also be inserted into an appropriate cloningvector for subsequent introduction into the genomes of suitable plantsthat are known to be infested with insects susceptible to thedelta-endotoxin(s), or into specific baculoviruses which can in turn bedirectly used as insecticides.

Those skilled in the art will recognize that the invention is notlimited to use of the nucleic acid fragments specifically disclosedherein, for example, in SEQ ID NO:39 OR 40. It will be apparent that theinvention also encompasses those nucleotide sequences that encode thesame amino acid sequences as depicted in SEQ ID NO:39 OR 40, but whichdiffer from those specifically depicted nucleotide sequences by virtueof the degeneracy of the genetic code. The invention specificallyencompasses any variant nucleotide sequence, and the protein encodedthereby, which protein retains at least about an 80%, preferably 90%,and most preferably 95% homology or identity with one or the other ofthe amino acid sequences depicted in FIG. 2 and retains the activity ofthe sequences described herein. In particular, variants which retain ahigh level (i.e., >80%) of homology at highly conserved regions of saiddelta-endotoxin are contemplated. Furthermore, the invention encompassesany variant that hybridizes to the nucleotide sequence of thedelta-endotoxin under the following conditions:presoaking in 5× SSC andprehydbridizing for 1 hr. at about 40° C. in a solution of 20%formamide, 5× Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and50 ug denatured sonicated calf thymus DNA, followed by hybridization inthe same solution supplemented with 100 uM ATP for 18 hrs. at about 40°C., followed by a wash in 0.4× SSC at a temperature of about 45° C.

Useful variants within the categories defined above include, forexample, ones in which conservative amino acid substitutions have beenmade, which substitutions do not significantly affect the activity ofthe protein. By conservative substitution is meant that amino acids ofthe same class may be substituted by any other of that class. Forexample, the nonpolar aliphatic residues Ala, Val, Leu, and Ile may beinterchanged, as may be the basic residues Lys and Arg, or the acidicresidues Asp and Glu. Similarly, Ser and Thr are conservativesubstitutions for each other, as are Asn and Gln. It will be apparent tothe skilled artisan that such substitutions can be made outside theregions critical to the function of the molecule and still result in anactive delta-endotoxin. Retention of the desired activity can readily bedetermined by using the assay procedures described below.

5.2. Mutants

The invention is also directed to a mutant B.t. strain which produces alarger amount of and/or larger crystals than the parental strain. A"parental strain" as defined herein is the original Bacillusthuringiensis strain before mutagenesis.

To obtain such mutants, the parental strain may, for example, be treatedwith a mutagen by chemical means such asN-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate, or byirradiation with gamma rays, X-rays or UV. Specifically, in one methodof mutating Bacillus thuringiensis strains and selecting such mutantsthe following procedure is used:

i) the parental strain is treated with a mutagen;

ii) the thus presumptive mutants are grown in a medium suitable for theselection of a mutant strain; and

iii) the mutant strain is selected for increased production ofdelta-endotoxin.

According to a preferred embodiment of this method, the selectedcolonies are grown in a production medium, and a final selection forstrains capable of increased delta-endotoxin production is performed.

Alternatively, the mutant(s) may be obtained using recombinant DNAmethods known in the art. For example, a DNA sequence containing a genecoding for a delta-endotoxin may be inserted into an appropriateexpression vector and subsequently introduced into the parental strainusing procedures known in the art. Alternatively, a DNA sequencecontaining a gene coding for a delta-endotoxin may be inserted into anappropriate vector for recombination into the genome and subsequentamplification.

5.3. Bioassay

The activity of the B.t. strains of the present invention or spores,mutants, crystals, or delta-endotoxins thereof against various insectpests may be assayed using procedures known in the art, such as anartificial insect diet incorporation assay, artificial diet overlay,leaf painting, leaf dip, and foliar spray. Specific examples of suchassays are given in Section 6, infra.

5.4. Compositions

The strains, spores, crystals, delta-endotoxins, or mutants of thepresent invention described supra can be formulated with an acceptablecarrier into an insecticidal composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol orimpregnated granule.

Such compositions disclosed above may be obtained by the addition of asurface active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a U.V. protectant, a buffer, a flow agent, or othercomponent to facilitate product handling and application for particulartarget pests.

Suitable surface-active agents include but are not limited to anioniccompounds such as a carboxylate, for example, a metal carboxylate of along chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulphates such as sodium dodecyl sulphate, sodiumoctadecyl sulphate or sodium cetyl sulphate; ethoxylated fatty alcoholsulphates; ethoxylated alkylphenol sulphates; lignin sulphonates;petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzenesulphonates or lower alkylnaphthalene sulphonates, e.g.butyl-naphthalene sulphonate; salts of sulphonatednaphthalene-formaldehyde condensates; salts of sulphonatedphenol-formaldehyde condensates; or more complex sulphonates such as theamide sulphonates, e.g. the sulphonated condensation product of oleicacid and N-methyl taurine or the dialkyl sulphosuccinates, e.g. thesodium sulphonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g. sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate;an oxygen-containing amine such as an amine oxide of polyoxyethylenealkylamine; an amide-linked amine prepared by the condensation of acarboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates or botanical materials such as wood products, cork, powderedcorncobs, peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate or primary powder which requiresdilution with a suitable quantity of water or other diluent beforeapplication. The insecticidal concentration will vary depending upon thenature of the particular formulation, specifically, whether it is aconcentrate or to be used directly. The composition contains 1 to 98% ofa solid or liquid inert carrier, and 0 to 50%, preferably 0.1 to 50% ofa surfactant. These compositions will be administered at the labeledrate for the commercial product, preferably about 0.01 lb-5.0 lb peracre when in dry form and at about 0.01 pts-10 pts per acre when inliquid form.

In a further embodiment, the strains, spores, crystals, delta-endotoxinsor mutants of the present invention can be treated prior to formulationto prolong the pesticidal activity when applied to the environment of atarget pest as long as the pretreatment is not deleterious to thecrystal delta-endotoxin. Such treatment can be by chemical and/orphysical means as long as the treatment does not deleteriously affectthe properties of the compositions. Examples of chemical reagentsinclude, but are not limited to, halogenating agents; aldehydes such asformaldehyde and glutaraldehyde; anti-infectives, such as zephiranchloride; alcohols, such as isopropranol and ethanol; and histologicalfixatives, such as Bouin's fixative and Helly's fixative (see, forexample, Humason, Animal Tissue Techniques, W. H. Freeman and Co.,1967).

The compositions of the invention can be applied directly to the plantby, for example, spraying or dusting at the time when the pest has begunto appear on the plant or before the appearance of pests as a protectivemeasure. Plants to be protected within the scope of the presentinvention include, but are not limited to, cereals (wheat, barley, rye,oats, rice, sorghum and related crops), beets (sugar beet and fodderbeet), drupes, pomes and soft fruit (apples, pears, plums, peaches,almonds, cherries, strawberries, raspberries, and blackberries),leguminous plants (alfalfa, beans, lentils, peas, soybeans), oil plants(rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants,cocoa beans, groundnuts), cucumber plants (cucumber, marrows, melons),fibre plants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons,grapefruit, mandarins), vegetables (spinach, lettuce, asparagus,cabbages and other brassicae, carrots, onions, tomatoes, potatoes,paprika), lauraceae (avocados, cinnamon, camphor), deciduous trees andconifers (e.g. linden-trees, yew-trees, oak-trees, alders, poplars,birch-trees, firs, larches, pines), or plants such as maize, turfplants, tobacco, nuts, coffee, sugar cane, tea, vines, hops, bananas andnatural rubber plants, as well as ornamentals. In most cases, thepreferred mode of application is by foliar spraying. The preferred modeof application for soil pests is by furrow application or by "lay-by"application. It is generally important to obtain good control of pestsin the early stages of plant growth as this is the time when the plantcan be most severely damaged. The spray or dust can conveniently containanother pesticide if this is thought necessary. In a preferredembodiment, the compositions of the invention is applied directly to theplant.

The compositions of the present invention may be effective against pestsincluding, but not limited to, pests of the order Lepidoptera, e.g.Achroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana,Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyeloistransitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria,Antheraea pernyi, Anticarsia gemmatalis, Archips sp., Argyrotaenia sp.,Athetis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella,Choristoneura sp., Cochylis hospes, Colias eurytheme, Corcyracephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima,Dendrolimus sibericus, Desmia funeralis, Diaphania hyalinata, Diaphanianitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomossubsignaria, Boreuma loftini, Ephestia elutella, Erannis tiliaria,Estigmene acrea, Eulia salubricola, Eupoecilia ambiguella, Euproctischrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta,Harrisina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothisvirescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantria cunea,Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdinafiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostegesticticalis, Lymantria dispar, Macalla thyrsisalis, Malacosoma sp.,Mamestra brassicae, Mamestra configurata, Manduca quinquamaculata,Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata,Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota sultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tineola bisselliella, Trichoplusia ni, Udearubigalis, Xylomyges curialis, Yponomeuta padella; Coleoptera, e.g.,Leptinotarsa sp., Acanthoscelides obtectus, Callosobruchus chinensis,Epilachna varivestis, Pyrrhalta luteola, Cylas formicarius elegantulus,Listronotus oregonensis, Sitophilus sp., Cyclocephala borealis,Cyclocephala immaculata, Macrodactylus subspinosus, Popillia japonica,Rhizotrogus majalis, Alphitobius diaperinus, Palorus ratzeburgi,Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum, Triboliumconfusum, Tribolius destructor.

In specific embodiments, a composition comprising the 130,000 daltondelta-endotoxins and/or the two 33,000 dalton delta-endotoxins iseffective against lepidopteran pests. Compositions comprising thestrains of the present invention are also effective against lepidopteranand coleopteran pests.

The following examples are presented by way of illustration, not by wayof limitation.

6. EXAMPLES 6.1.Example 1 Cultivating B.t. Strains EMCC0075 and EMCC0076

Subcultures of EMCC0075 and EMCC0076, maintained on Nutrient Broth Agarslants, are used to inoculate 250 ml baffled shake flasks containing 50ml of medium with the following composition:

Corn Steep liquor 15 g/l

Maltrin-100 40 g/l

Potato Starch 30 g/l

KH₂ PO₄ 1.77 g/l

K₂ HPO₄ 4.53 g/l

The pH of the medium is adjusted to 7.0 using 10N NaOH.

After inoculation, shake flasks are incubated at 30° C. on a rotaryshaker with 250 rpm shaking for 72 hours. The B.t. crystals and spores,obtained in the above fermentation, are recovered by centrifugation at15,000 rpm for 15 minutes using a Sorvall RC-5B centrifuge.

6.2. Example 2 Testing of B.t. Strains EMCC0075 and EMCC0076 Spores andCrystals

EMCC0075 and EMCC0076 are cultivated in shake flasks as described inExample 1, supra. To determine if EMCC0075 and EMCC0076 are activeagainst lepidopteran pests, a 1:50 dilution of culture broth is made. 5ml of such diluted culture broth is transferred into a 50 mlpolypropylene centrifuge tube. 20 ml of artificial insect dietcontaining antibiotics is added into the centrifuge tube. The mixture issubsequently dispensed into bioassay trays. Three to six eggs either ofbeet armyworm (Spodoptera exigua) or tobacco budworm (Heliothisvirescens) are applied on the surface of the "diet". Mylar is ironedonto the bioassay trays and the trays are incubated at 28° C. Scoring iscarried out at 7 and 11 days.

To determine if EMCC0075 and EMCC0076 are active against insect pests ofthe order Coleoptera, 5 ml of the culture broths are removed from theshake flasks and transferred directly into the 50 ml polypropylenecentrifuge tubes. 20 ml of artificial insect diet (containing knownantibiotics) are then added into the tubes (final testingconcentration=20% w/w) and mixed vigorously. The mixtures are thendispensed into bioassay trays. Three to six eggs of corn rootworm(Diabrotica undecimpunctata) are applied to the surface of the "diet".Mylar is ironed onto the bioassay trays and the trays are incubated at28° C. Scoring is carried out at 7 and 11 days.

The bioactivity of EMCC0075 and EMCC0076 towards Spodoptera exigua andDiabrotica undecimpunctata is expressed in terms of stunt score (SS).The stunt score is determined after incubating the trays for 7 days. Inthis system, 4=full size larvae (control larvae); 3=3/4 size of controllarvae; 2=1/2 size of control larvae; 1=1/4 size of control larvae; and0=mortality. The smaller the number, the higher the B.t. activity. Theresults are shown in Table I. It is evident that EMCC0075 and EMCC0076possess activity against both lepidopteran and coleopteran pests.

                  TABLE I    ______________________________________            Spodoptera                      Diabrotica  Heliothis            exigua    undecimpunctata                                  virescens    ______________________________________    EMCC0075  1.7         0.9         1.5    EMCC0076  1.8         1.8         1.8    Control   4.0         4.0         4.0    ______________________________________

6.3. Example 3 cry Gene Profile for EMCC0075 and EMCC0076

The cry gene profile for EMCC0075 and EMCC0076 is determined by usingthe PCR method which is described in the Perkin Elmer Cetus Gene Amp®PCR Reagent Kit literature. Double-stranded DNA is heat-denatured andthe two oligonucleotides corresponding to the cryIA(a) gene (listed inthe Sequence Listing as SEQ ID NO:3 and SEQ ID NO:4 respectively),cryIA(b) gene (listed in the Sequence Listing as SEQ ID NO:5 and SEQ IDNO:6 respectively), cryIA c) gene (listed in the Sequence Listing as SEQID NO:7 and SEQ ID NO:8 respectively), cryID gene (listed in theSequence Listing as SEQ ID NO:9 and SEQ ID NO:10 respectively), cryIIIAgene (listed in the Sequence Listing as SEQ ID NO:l1 and SEQ ID NO:12respectively), cryIIIB gene (listed in the Sequence Listing as SEQ IDNO:13 and SEQ ID NO:14 respectively), cryIIIc gene (listed in theSequence Listing as SEQ ID NO:15 and SEQ ID NO:16 respectively), andcryIIID gene (listed in the Sequence Listing as SEQ ID NO:17 and SEQ IDNO:18 respectively), are annealed at low temperature and then extendedat an intermediate temperature.

PCR analysis indicated that both strains contain a cryID-like gene. Aprobe specific to cryID also detected a cryID-like gene in Southernanalysis of restricted genomic DNA from both strains. No PCRamplifications are observed with primers to cryIA(a), cryIA(b),cryIA(c), cryIB (SEQ ID NOS:22 and 23), cryIC (SEQ ID NOS:24 and 25),cryID, cryIE (SEQ ID NOS:26 and 27), cryIF (SEQ ID NOS:28 and 29), orcryIG (SEQ ID NOS:30 and 31), nor to cryIIA (SEQ ID NOS:32 and 33),cryIIB (SEQ ID NOS:34 and 33), or cryIIC (SEQ ID NOS: 35 and 36), nor tocryIIIA, cryIIIB, cryIIIC, or cryIIID. However, Southern analysis of arestriction fragment from genomic DNA from EMCC0075 and EMCC0076 with aprobe that can detect cryIA(a), cryIA(b), and cxyIA(c) confirmed thepresence of a cryIA-like gene.

6.4. Example 4 Purification of EMCC0075 Bipyramidal and RhomboidalCrystals

A subculture of EMCC0075, maintained on a Nutrient Broth agar plate, isused to inoculate a 2.0 liter baffled shake flask containing 500 ml ofmedium with the same composition as described in Example 5, infra. Afterinoculation, the shake flask is incubated at 30° C. on a rotary shakerfor 72 hours at 250 rpm. The crystals and spores are recovered bycentrifugation at 10,000 rpm (Sorvall GSA rotor) for 30 minutes. Thepellets are washed with deionized water, centrifuged at 15,000 rpm(Sorvall SS34 rotor), and resuspended in deionized water by sonicationto a concentration of 0.1 g wet weight per ml. 1 g wet weight crudecrystals are diluted to 33.2 ml with deionized water and placed in a 250ml separatory funnel. The bottom phase solution comprised of 10 ml 3Msodium chloride, 23.4 ml 20% polyethylene glycol 8000, and 33.4 ml 20%sodium dextran sulfate is added to the 250 ml separatory funnel andmixed, followed by 100 ml of a polyethylene glycol upper phase solutioncomprised of 0.3 g sodium dextran sulfate, 70.3 g polyethylene glycol8000, and 17.5 g sodium chloride per liter deionized water. Thesuspension is shaken vigorously, and the two phases are allowed toseparate at room temperature for 30 minutes.

The upper phase which contains large quantities of spores is removedwith a pipet. The lower phase contains crystals and residual spores. Theextraction is repeated several times until the upper phase containsessentially no spores. The lower phase is then diluted with 100 mldeionized water, and centrifuged at 10,000 rpm (Sorvall GSA rotor) for45 minutes at 5° C. to recover the crystals. The recovered crystals arewashed with 200 ml deionized water, and recentrifuged as before. Thespores from the upper phase are also recovered using the above washingprocedure.

The bipyramidal and rhomboidal crystals are then further purified bydensity gradient centrifugation using a discontinuous LudoxTM HS-40(DuPont) gradient comprised of 3.8 ml each of 75%, 50%, and 38% Ludox™v/v adjusted to pH 2.5 with 0.2M Tris-HCl. 10 mg of crystals in 100 μldeionized water are layered on the top of the gradient, and centrifugedin a Beckman Ultracentrifuge at 10,000 rpm (Beckman 41 Ti rotor) for 15minutes at 20° C. Four separate bands are obtained. One contains purerhomboidal crystals and another contains pure bipyramidal crystals. Thetwo other bands contains mixtures of the two crystal types. The purecrystal bands are recovered, washed with deionized water, and used forbioassay.

6.5. Example 5 SDS-Page Analysis of the Delta-Endotoxins from EMCC0075and EMCC0076

Subcultures of EMCC0075 and EMCC0076, maintained on Nutrient Broth agarplates, are used to inoculate 250 ml baffled shake flasks containing 50ml of medium with the following composition:

Glucose 2.0 g/l

KH₂ PO₄ 0.86 g/l

K₂ HPO₄ 0.55 g/l

Sodium Citrate 2.0 g/l

CaCl₂ 0.1 g/l

MnCl₂.4H₂ O 0.16 g/l

MgCl₂.6H₂ O 0.43 g/l

ZnCl₂ 0.007 g/l

FeCl₃ 0.003 g/l

Casamino Acids 5 g/l

After inoculation, the shake flasks are incubated at 30° C. on a rotaryshaker for 72 hours at 250 rpm. The B.t. crystals obtained in the abovefermentations of EMCC0075 and EMCC0076 are recovered by centrifugationat 10,000 rpm (Sorvall GSA rotor) for 30 minutes. The B.t. crystals arethen purified by biphasic extraction using sodium dextran sulfate andpolyethylene glycol as outlined in Example 4, supra.

B.t. crystal preparations from EMCC0075 and EMCC0076 are analyzed bySDS-PAGE. Specifically, the SDS-PAGE is carried out on 10-15% gradientgels using Pharmacia's Phast System. The protein bands are analyzed on aPharmacia densitometer using Pharmacia Gelscan™ Software. The resultsindicated that the crystals produced by both strains contain at leasttwo proteins with molecular weights of approximately 130,000 daltons and33,000 daltons.

6.6. Example 6 Bioassay Using Spodoptera Exigua to Determine Activity ofNovel Lepidopteran Active Bacillus thuringiensis Strains

To determine if purified bipyramidal and rhomboidal crystals are activeagainst lepidopteran pests, the crystals are bioassayed againstSpodoptera exigua using a surface overlay assay. Samples of crystalpreparations are applied to individual wells of a jelly tray containing500 μl of solidified artificial insect diet per well. The trayscontaining the various samples are air dried. Two to four 2nd or early3rd instar Spodoptera exigua are added to each well containing the driedtest sample. The trays are then sealed with Mylar punched with holes forair exchange and are incubated for 3 days at 30° C. The degree ofstunting, as described in Example 2, supra, is then recorded.

The results are shown in Table II. It is evident that, surprisingly,both the bipyramidal crystal and the rhomboidal crystal possess activityagainst Spodoptera exigua. The spores also show activity againstSpodoptera exigua.

                  TABLE II    ______________________________________    Sample           Wet Weight  Stunt score    ______________________________________    No crystals or spores                     --          4    Rhomboidal & bipyramidal                     2.5 mg/well 1    crystals and spores                     5.0 mg/well 0-1    Both crystals, no spores                     2.5 mg/well 1                     10 mg/well  0-1    Bipyramidal crystals                     0.092 mg/well                                 1                     0.48 mg/well                                 0-1    Rhomboidal crystals                     0.05 mg/well                                 1                     0.1 mg/well 0-1                     0.5 mg/well 0    Spores           10 mg/well  0-1                     20 mg/well  0    ______________________________________

6.7. Example 7 Bioassay Against Diabrotica undecimpunctata

The coleopteran activity of the whole culture broth of EMCC0075,prepared as described in EXAMPLE 1, is bioassayed against Diabroticaundecimpunctata using a micro-diet incorporation bioassay. Specifically,artificial diet is prepared comprised of water, agar, sugar, casein,wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts,propionic acid, phosphoric acid, streptomycin, chlortetracycline, andvitamins. The artificial diet is developed to allow samples consistingof rehydrated dry powders and liquids to be incorporated at a rate of20% v/v. The test sample is prepared in microcentrifuge tubes to yieldeight serial dilutions. The whole broth sample is tested neat at 200μl/ml, and then diluted in 0.1% Tween 20 to contain 132 μl/ml, 87 μl/ml,66 μl/ml, 44 μl/ml, 30 μl/ml, 20 μl/ml, and 13 μl/ml. The molten mixtureis vortexed and pipetted in 0.1 ml aliquots into 10 wells of a 96 wellmicrotiter plate. Control samples containing 0.1% Tween 20 are dsipensedinto 16 wells. Once the diet has cooled and solidified, two neonateDiabrotica undecimpunctata larvae are added to each well, and the traysare covered with a perforated sheet of clear mylar. The trays are thenincubated for five days at 28°±2° C. and 65% relative humidity.

After five days, insect mortality is rated. The mylar sheet is removedand each well of the microtiter plate is inspected using a dissectingmicroscope. Larvae that do not move when prodded with a dissectingneedle are counted as dead. Percent mortality is calculated, and thedata is analyzed via parallel probit analysis. The LC₅₀, LC₉₀, slope ofregression lines, coefficient of variation (CV), and potencies aredetermined.

The results as shown in Table III indicate the whole culture broth fromEMCC-0075 has a LC₅₀ and a LC₉₀ of 51 μl/ml diet and 170 μl/ml diet,respectively, against Diabrotica undecimpunctata.

                  TABLE III    ______________________________________    LC.sub.50  LC.sub.90                      Slope        CV  N    ______________________________________    μl/ml   μl/ml    51         170    2            7   8    ______________________________________

6.8. Example 8 Protein Sequencing of the Delta-Endotoxins from theRhomboidal Crystal Proteins of EMCC0075

60 μl of 50% trifluoroacetic acid (TFA) are added to 25 μg of rhomboidalcrystals. Four 15 μl aliquots of the mixture are spot dried onto aBiobrene-coated and TFA-pretreated microcartridge glass fiber filter.N-terminal sequencing is performed on a Applied Biosystems Inc. ProteinSequencer Model 476A with on-line HPLC and liquid phase TFA delivery.HPLC determination of phenylthiohydantoin-amino acids is achieved byusing the Premix buffer system (ABI Inc.). Data is collected on aMacintosh IIsi using ABI's 610 data analysis software.

A double sequence is observed at approximately a 60/40 ratio. Data areanalyzed and the sequences are sorted as follows:

    "MIVDL": MIVDLYRYLGGLAAVNAVLHFYEPRP                        (SEQ ID NO:1)

    "MKHHK": MKHHKNFDHI                                        (SEQ ID NO:2)

6.9. Example 9 Cloning of the Genes Endoding The "MIVDL" and "MKHHK"Proteins

The amino acid sequence initially determined for the "MIVDL" protein,MIVDLYRYLGGLAAVNAVLHFYEPRP, is encoded by the sequence ATG ATH GTN GAYYTN TAY MGN TAY YTN GGN GGN YTN GCN GCN GTN AAY GCN GTN YTN CAY TTY TAYGAR CCN MGN CCN (SEQ ID NO:19). Based on this sequence, a 71 nt oligomeris designed, where mixed deoxynucleotides are used at the 2-foldredundant positions and deoxyinosine at the 4-fold redundant positionsto decrease both base discrimination at mismatches and selectivity atincorrect bases (Martin, F. H., and M. M. Castro, 1985, Nucleic AcidsRes. 13: 892-8938): ATG ATI GTI GAY YTI TAY MGI TAY YTI GGI GGI YTI GCIGCI GTI AAY GCI GTI YTI CAY TTY TAY GAR CC (SEQ ID NO:20).

The amino acid sequence determined for the "MKHHK" protein, namely,MKHHKNFDHI, permitted design of a more discriminating probe because ofthe absence of amino acids specified by more than two codons. Furtherdiscrimination is permitted by the assumption that As or Ts would beused in the coding sequence in preference to Gs or Cs, due to theoverall low % G+C content of B. t. strains (approx 34 moles %, Claus,D., and R. C. W. Berkeley. 1986. Genus Bacillus, p. 1112. In P. H. A.Sneath (ed.), Bergey's manual of systematic bacteriology, v. 2. TheWilliams and Wilkins Co., Baltimore). The following probe issynthesized: ATG AAA CAT AAA AAT TTT GAT CAT AT (SEQ ID NO:21). Both theMIVDL and the MKHHK probes are tailed with digoxygenin-dUTP according tothe manufacturer's instructions (Boerhinger-Mannheim Genius System™Users Guide, Version 2.0).

EMCC0075 genomic DNA is digested with EcoRI, EcoRV, HindIII, PstI, orcombinations of those enzymes overnight in buffers supplied by themanufacturers, electrophoresed through 0.8% agarose in 0.5× TBE(TRIS-borate-EDTA buffer; Sambrook et al., 1989, in Molecular Cloning, aLaboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,N.Y.), transferred in 10× SSC to Boehringer Mannheim nylon membrane witha Stratagene Posiblotter in 10× SSC, and then probed as described below.The MIVDL probe, after hybridization and stringent washing at 48° C.with 0.5× SSC, detected EcoRV and PstI fragments 12 kb or more in size,an EcoRI fragment of approx 10 kb, and a HindIII fragment of approx 3.5kb. The MKHHK probe, after hybridization and stringent washing at 48° C.with 5× SSC, detected the same size EcoRI, EcoRV, and PstI fragments asdid the MIVDL probe. This result indicates that the two genes lie inclose proximity to each other. Additionally, the MKHHK probe detected aHindIII fragment of approx 6 kb.

To clone the HindIII fragments encoding at least part of the "MIVDL" and"MKHHK" proteins, pUC118 is digested with HindIII, and then treated withcalf intestinal phosphatase to dephosphorylate the 5' ends and thusprevent vector religation. Restricted and phosphatased pUC118 is thenmixed with EMCC0075 genomic DNA that had been previously digested tocompletion with HindIII. After ligation, the reaction mix is used totransform E. coli strain XL1-Blue MRF' (Stratagene, Inc., La Jolla,Calif.). Colonies harboring the desired DNA fragment are detected by"colony hybridization" with the aforementioned "MIVDL" and "MKHHK"probes by the procedure described by Sambrook et al., 1989, Molecularcloning, A Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. Three fragments are cloned with the "MIVDL" and "MKHHK"probes (see FIG. 2). E. coli containing the "13D" MIVDL gene fragmentaew referred to as EMCC0117 cells; E. coli containing the "8D-1" MKHHKgene fragment are referred to as EMCCO118 cells; E. coli containing the"2B" fragment of the MIVDL and MKHHK genes are referred to as EMCCO118cells.

6.10. Example 10 Sequencing of the Genes Encoding the "MIDVDL" and"MKHHK" Proteins

Nested deletions of three cloned fragments described in EXAMPLE 9 areperformed according to the method of Henikoff (Gene 28: 351-359, 1984)with a Promega "Erase-a-Base" kit. Nested deletion sets encompassing theregion of interest are sequenced by the dideoxy method (Sanger et al.,1977, PNAS USA 74: 5463-5467) with an ABI 373A sequencer. Sequencecorrection is performed with SeqEd v 1.0.3; sequence is assembled withMacvector 4.1.1 and AssemblyLIGN v 1.0.7; and additional alignments andsearches are performed with the IntelliGenetics Suite Programs, v 5.4.

The determined nucleotide (nt) sequence encoding the MKHHK and MIVDLproteins are shown in SEQ ID NO:39 and 40. The deduced amino acidsequence of the MKHHK and MIVDL proteins is shown underneath theircorresponding DNA sequence. The amino acid sequence determined byN-terminal Edman degradation as described in EXAMPLE 8 is in completeagreement with the sequences deduced from the nucleotide sequence. Thegenomic DNA sequence is shown in SEQ ID NOS:41 (MKHHK and MIVDL), 44(MKHHK), and 45 (MIVDL).

The MKHHK and MIVDL genes encode proteins with calculated molecularmasses of 32,719 and 32,866 daltons. The MKHHK protein aligns poorlywith any deduced protein from the EMBL, GeneSeq, or GenBank sequencedatabases. The MIVDL protein has weak regional homology with the 34 kdalgene of B. thuringiensis subsp. thompsoni as shown in FIG. 3 (SEQ IDNO:42) (Brown and Whiteley, 1990, J. Bacteriology 174: 549-557). Inaddition, the MIVDL protein has weak regional homologies with CryIA(a)(SEQ ID NO:43) (see FIG. 3). These weak homologies do not correspond tothe any of the 5 conserved blocks of Cry toxins described by Hofte andWhiteley (Microbiol. Rev. 53: 242-255, 1989).

A nucleotide analysis of the region encoding the MKHHK and MIVDL genesshows ribosome binding sites (AAGGAGT and AAGGTGG, respectively) thatdiffer by one nucleotide with the canonical ribosome binding site of B.subtilis (AAGGAGG, which is presumably similar to the B. thuringiensisRBS). There is a reasonable transcriptional terminator downstream of theMIVDL gene.

7. DEPOSIT OF MICROORGANISMS

The following strains of Bacillus thuringiensis have been deposited inthe Agricultural Research Service Patent Culture Collection Laboratory(NRRL), Northern Regional Research Center, 1815 University Street,Peoria, Ill., 61604, USA.

    ______________________________________    Strain      Accession Number                              Deposit Date    ______________________________________    EMCC0075    NRRL B-21019  December 3, 1992    EMCC0076    NRRL B-21020  December 3, 1992    ______________________________________

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122 and under conditions of the Budapest Treaty. The deposit representsa biologically pure culture of each deposited strain. The deposit isavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 45    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetIleValAspLeuTyrArgTyrLeuGlyGlyLeuAlaAlaValAsn    151015    AlaValLeuHisPheTyrGluProArgPro    2025    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetLysHisHisLysAsnPheAspHisIle    1510    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CTGCTCCAGCTGCTTGGCTC20    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GAATTATACTTGGTTCAGGCCC22    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GCACACCTTACATTTTAAAGCA22    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AGATTACAAGCGGATACCAACATCGCG27    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    TGGCACTTTCAAAATAACCAA21    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GCATCGGATAGTATTACTCAAATCCC26    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    CGCTCTAACATAGACCTTATAA22    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GACATTTCATTAGGGCTTATTAATTT26    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    CAGCGGACGGCCAGACCGCAAG22    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GTCGGAGTCAACAACCTTAGGGGC24    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    ATCCGGAAAAGCCGCTATGTC21    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    ATCCGGAAAAGCCGCTATGTC21    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GGCCAGAAAATGGAAAAATTTGGG24    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GTGGGTACAGGAGGTACCAAA21    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GTGGGTACAGGAGGTACCAAA21    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    CGAAATACTATGAGTGTAACTGC23    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 54 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    YTNGGNGGNYTNGCNGCNGTNAAYGCNGTNYTNCAYTTYTAYGARCCNMGNCCN54    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 57 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    ATGATGTGAYYTTAYMGTAYYTGGGGYTGCGCGTAAYGCGTYTCAYTTYTAYGARCC57    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    ATGAAACATCATAAAAATTTTGATCATAT29    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    TTGAATTCATATCTACTAATGAGCAATCGAA31    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    CCACACGCCTAGATTCTCATGC22    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 46 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    CGGGATCCACAGTTACAGTCTGTAGCTCAATTACCTACTTTTAACG46    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    GGCCAAGGTTGCTGTAATAATCG23    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    CTCAATATTCTCGAAGCTGGGGCC24    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GCAGTCTGTACGGAATTTATACA23    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    CGAGGGTTAGCAGATAGCTATG22    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    AAGATGGGGCGGTCTAACTCC21    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GACCGTTATCGGGTGAATCTTTAG24    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    TCGGCTGCACTCTAAATTGTTGAG24    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    TATTGAGTGAATTATGGGGGAT22    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    ATGTTCTAAATTCTAACATATCG23    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    TTATACCTAGATCCTATTGTTG22    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    TAACATTTCCACACTTTTCAATC23    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    AAGGCTAGCGACTGCTGTC19    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 287 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    MetLysHisHisLysAsnPheAspHisIleValTrpAspPheAlaGlu    151015    LysTrpThrGluGlnLysGlyValAspLeuLysArgValSerTyrVal    202530    AspProIleThrGlyGluAspThrLeuGluPheIleThrLysPheAsn    354045    TyrValGlyLysLeuGluGluLysAlaTyrCysProGluValIleGlu    505560    ThrGlnSerPheSerAsnSerAsnCysAspValSerArgGluPheLeu    65707580    LysLysLysValAspArgLysGluCysTyrLeuTrpAspIleAspTyr    859095    GlyPheIleIleProThrSerValLeuThrAsnProLeuLeuProPro    100105110    ThrLeuAsnGluLysIleAsnProAlaMetGluValAspLeuPheLys    115120125    SerAlaAsnLeuPheGluSerLysLeuAsnAsnTyrArgMetIleGlu    130135140    AlaGlyValTyrIleGluProAsnGlnAlaValThrAlaSerIleMet    145150155160    ValThrProLysGlnValGlnGlnAspTyrCysIleSerLeuGluIle    165170175    SerGlySerIleIleIleGluLeuLysAspAlaTyrAsnAlaCysThr    180185190    AspLysGluThrIleGluThrIlePheTyrThrValProIleAlaAsp    195200205    IleTyrArgSerGluLeuAlaHisAsnHisSerPheHisLeuAspGly    210215220    GluThrValIlePheThrGlyLysGlyThrPheLysGlyLeuIleCys    225230235240    SerAsnIlePheValGluGlyGluArgPheAspSerGlnThrGlyGlu    245250255    CysLeuGlyLysTyrValIleProLeuSerIleGluLysLysAsnAsn    260265270    ValAspCysIleSerIlePheLeuAsnSerGluLysGlyGlyIle    275280285    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 294 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    MetIleValAspLeuTyrArgTyrLeuGlyGlyLeuAlaAlaValAsn    151015    AlaValLeuHisPheTyrGluProArgProAspIleCysArgAsnIle    202530    SerGluGluTyrAsnLeuIleValPheGlyAspArgIleProThrPhe    354045    SerIleAspProSerGlnIleAsnIleAsnAsnLeuSerValAspThr    505560    ProValAspGluIleThrIleAsnAsnValArgSerIleGlnLeuIle    65707580    SerSerArgPheGluAsnThrGlyPheValAspThrGluAsnTyrPhe    859095    ThrProGluLeuSerArgThrValValAsnSerIleSerThrSerThr    100105110    ThrThrGlyTyrLysTyrThrGlnSerLeuThrValSerSerLysPhe    115120125    SerPheAsnPheProValAlaGlyAlaGluAsnAsnIleSerPheSer    130135140    ValGlyPheGluGlnAsnLeuSerThrThrGluThrLysThrGluSer    145150155160    ThrSerThrLeuMetArgIleProProGlnProValSerValArgPro    165170175    ArgThrAlaLysArgValGluIleSerLeuPheGluLeuAlaIlePro    180185190    ArgIleGlnAsnGluIleSerGlyPheValThrGlyThrLeuProThr    195200205    IleSerAsnSerHisIleSerAspLeuTyrAlaValLeuThrArgThr    210215220    AspSerLeuCysProAsnSerTyrIleAsnArgAspAspPheLeuArg    225230235240    IleAspHisGluAsnArgGlyLeuGlyLeuGlnGlyPheGlySerLeu    245250255    ThrGlyAsnLeuThrSerLeuAspPheAlaIleArgThrThrGluTyr    260265270    AspLeuProSerAsnThrIleIleAsnIleGluAsnGluIleLysArg    275280285    AlaHisIleLeuThrGln    290    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 864 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    ATGAAACATCATAAAAATTTTGATCACATAGTTTGGGACTTCGCTGAAAAGTGGACTGAA60    CAAAAGGGGGTAGATTTAAAAAGGGTCAGTTATGTAGATCCCATTACTGGTGAAGATACA120    TTAGAGTTTATAACCAAATTTAATTATGTTGGGAAATTAGAAGAAAAAGCTTATTGTCCA180    GAAGTAATAGAAACACAATCTTTTTCAAACTCAAATTGTGACGTTTCGAGGGAATTTCTA240    AAGAAAAAAGTAGACAGGAAGGAATGTTATTTATGGGATATAGACTATGGGTTTATTATA300    CCAACTTCGGTACTTACAAATCCATTATTACCCCCCACTCTCAATGAAAAAATTAATCCA360    GCAATGGAAGTGGACTTATTTAAAAGTGCAAACCTGTTTGAATCCAAACTAAATAATTAT420    AGAATGATAGAAGCAGGTGTTTATATTGAACCAAATCAAGCAGTAACCGCCAGCATAATG480    GTTACACCAAAACAAGTACAGCAAGATTATTGTATTAGCCTTGAGATTTCAGGTAGTATT540    ATCATTGAGCTGAAAGATGCTTATAATGCTTGTACAGATAAAGAAACTATTGAAACAATA600    TTCTATACCGTGCCAATTGCAGATATATACAGATCCGAGCTTGCCCATAACCATTCCTTT660    CATTTAGATGGAGAAACTGTAATATTTACAGGGAAAGGTACGTTTAAAGGCTTAATATGT720    TCTAATATATTTGTTGAAGGGGAAAGATTCGATTCTCAAACGGGGGAATGTTTGGGGAAA780    TATGTGATCCCATTAAGTATAGAAAAGAAAAATAATGTAGATTGTATCTCTATATTTTTA840    AATTCAGAAAAAGGTGGGATTTAA864    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 885 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    ATGATAGTAGATTTATATAGATATTTAGGTGGATTGGCAGCAGTAAATGCCGTACTTCAC60    TTTTATGAGCCACGCCCTGATATATGTAGGAATATAAGCGAAGAATATAACCTTATAGTA120    TTTGGAGACCGTATACCAACTTTTAGCATAGATCCTTCGCAAATAAATATTAACAATTTA180    TCTGTGGACACTCCAGTGGATGAAATAACTATTAATAACGTGAGAAGTATACAATTAATA240    TCTAGTCGTTTTGAAAATACAGGATTTGTCGATACTGAAAATTATTTTACTCCTGAATTA300    TCTAGAACAGTTGTAAATAGCATATCTACATCGACTACTACAGGATATAAGTACACTCAA360    TCCCTTACTGTTTCATCCAAATTCTCCTTTAATTTCCCAGTTGCGGGTGCAGAAAATAAT420    ATTTCATTTTCAGTAGGTTTTGAACAAAACCTTTCAACTACAGAAACTAAAACAGAAAGT480    ACTTCAACGCTTATGCGTATACCTCCACAACCAGTTTCCGTAAGACCCAGAACAGCAAAA540    AGGGTTGAAATATCGCTCTTTGAATTGGCAATCCCTAGAATACAAAACGAAATTTCCGGA600    TTTGTAACAGGTACTCTTCCAACAATTTCAAATTCGCATATTTCCGATCTTTATGCTGTA660    TTAACACGGACTGATAGCCTATGCCCTAATTCATATATTAACCGAGATGACTTTTTAAGA720    ATAGATCATGAAAATAGGGGTTTGGGATTACAAGGCTTCGGTTCTCTCACTGGAAATTTA780    ACATCATTAGATTTTGCAATTAGAACTACTGAATATGATTTACCTTCAAATACAATTATA840    AATATAGAGAACGAAATAAAAAGAGCCCATATACTCACACAGTAA885    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2101 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    ATTAAACACTAAATACATTCACATTATTCTAACAAAGAAAAGGAGTAATAATTATGAAAC60    ATCATAAAAATTTTGATCACATAGTTTGGGACTTCGCTGAAAAGTGGACTGAACAAAAGG120    GGGTAGATTTAAAAAGGGTCAGTTATGTAGATCCCATTACTGGTGAAGATACATTAGAGT180    TTATAACCAAATTTAATTATGTTGGGAAATTAGAAGAAAAAGCTTATTGTCCAGAAGTAA240    TAGAAACACAATCTTTTTCAAACTCAAATTGTGACGTTTCGAGGGAATTTCTAAAGAAAA300    AAGTAGACAGGAAGGAATGTTATTTATGGGATATAGACTATGGGTTTATTATACCAACTT360    CGGTACTTACAAATCCATTATTACCCCCCACTCTCAATGAAAAAATTAATCCAGCAATGG420    AAGTGGACTTATTTAAAAGTGCAAACCTGTTTGAATCCAAACTAAATAATTATAGAATGA480    TAGAAGCAGGTGTTTATATTGAACCAAATCAAGCAGTAACCGCCAGCATAATGGTTACAC540    CAAAACAAGTACAGCAAGATTATTGTATTAGCCTTGAGATTTCAGGTAGTATTATCATTG600    AGCTGAAAGATGCTTATAATGCTTGTACAGATAAAGAAACTATTGAAACAATATTCTATA660    CCGTGCCAATTGCAGATATATACAGATCCGAGCTTGCCCATAACCATTCCTTTCATTTAG720    ATGGAGAAACTGTAATATTTACAGGGAAAGGTACGTTTAAAGGCTTAATATGTTCTAATA780    TATTTGTTGAAGGGGAAAGATTCGATTCTCAAACGGGGGAATGTTTGGGGAAATATGTGA840    TCCCATTAAGTATAGAAAAGAAAAATAATGTAGATTGTATCTCTATATTTTTAAATTCAG900    AAAAAGGTGGGATTTAACATGATAGTAGATTTATATAGATATTTAGGTGGATTGGCAGCA960    GTAAATGCCGTACTTCACTTGATTTAAACATGATAGTAGATTTATATAGATATTTAGGTG1020    GATTGGCAGCAGTAAATGCCGTACTTCACTTTTATGAGCCACGCCCTGATATATGTAGGA1080    ATATAAGCGAAGAATATAACCTTATAGTATTTGGAGACCGTATACCAACTTTTAGCATAG1140    ATCCTTCGCAAATAAATATTAACAATTTATCTGTGGACACTCCAGTGGATGAAATAACTA1200    TTAATAACGTGAGAAGTATACAATTAATATCTAGTCGTTTTGAAAATACAGGATTTGTCG1260    ATACTGAAAATTATTTTACTCCTGAATTATCTAGAACAGTTGTAAATAGCATATCTACAT1320    CGACTACTACAGGATATAAGTACACTCAATCCCTTACTGTTTCATCCAAATTCTCCTTTA1380    ATTTCCCAGTTGCGGGTGCAGAAAATAATATTTCATTTTCAGTAGGTTTTGAACAAAACC1440    TTTCAACTACAGAAACTAAAACAGAAAGTACTTCAACGCTTATGCGTATACCTCCACAAC1500    CAGTTTCCGTAAGACCCAGAACAGCAAAAAGGGTTGAAATATCGCTCTTTGAATTGGCAA1560    TCCCTAGAATACAAAACGAAATTTCCGGATTTGTAACAGGTACTCTTCCAACAATTTCAA1620    ATTCGCATATTTCCGATCTTTATGCTGTATTAACACGGACTGATAGCCTATGCCCTAATT1680    CATATATTAACCGAGATGACTTTTTAAGAATAGATCATGAAAATAGGGGTTTGGGATTAC1740    AAGGCTTCGGTTCTCTCACTGGAAATTTAACATCATTAGATTTTGCAATTAGAACTACTG1800    AATATGATTTACCTTCAAATACAATTATAAATATAGAGAACGAAATAAAAAGAGCCCATA1860    TACTCACACAGTAATTAATAGAAATAGACCGATAATCGGTCTTCCCCCTGTCAAGTAGGC1920    CTAGTGACAGGGTTCTTGCTGTGGACCGCAAGGTAGCAAATTTCTGAAGACCCATATGGG1980    GTACCGTCAGGAAAATGCGGATTTACAACGCTAAGCCCATTTTCCTGACGATTCCCCCAT2040    TTTTAACAACGTTAAGAAAGTTTCAATGGTCTTAAAGAATCTAATGAGATCATTTTCTCC2100    G2101    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 310 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    MetAlaIleMetAsnProArgProAspIleAlaGlnAspAlaAlaArg    151015    AlaTrpAspIleIleAlaGlyProPheIleArgProGlyThrThrPro    202530    ThrAsnArgGlnLeuPheAsnTyrGlnIleGlyAsnIleGluValGlu    354045    ThrProProGlyAsnLeuAsnPheSerValValProGluLeuAspPhe    505560    SerValSerGlnAspLeuPheAsnAsnThrSerValGlnGlnSerGln    65707580    ThrTyrAlaSerPheAsnGluSerArgThrValValGluThrThrSer    859095    ThrAlaValThrHisGlyValLysSerGlyValThrValSerAlaSer    100105110    AlaLysPheAsnAlaLysIleLeuValLysSerIleGluGlnThrIle    115120125    ThrThrThrValSerThrGluTyrAsnPheSerSerThrThrThrArg    130135140    ThrAsnThrValThrArgGlyTrpSerIleProAlaGlnProValLeu    145150155160    ValProProHisSerArgValThrAlaThrLeuGlnIleTyrLysGly    165170175    AspPheThrValProValLeuGlnAsnGluLeuSerLeuArgValTyr    180185190    GlyGlnThrGlyThrLeuProAlaGlyAsnProSerPheProSerAsp    195200205    LeuTyrAlaValAlaThrTyrGluAsnThrLeuLeuGlyArgIleArg    210215220    GluHisIleAlaProProAlaLeuPheArgAlaSerAsnAlaTyrIle    225230235240    SerAsnGlyValGlnAlaIleTrpArgGlyThrAlaThrThrArgVal    245250255    SerGlnGlyLeuTyrSerValValArgIleAspGluArgProLeuAla    260265270    GlyTyrSerGlyGluThrArgThrGluTyrTyrLeuProValThrLeu    275280285    SerAsnSerSerGlnIleLeuThrProGlySerLeuGlySerGluIle    290295300    ProIleIleAsnProVal    305310    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 358 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    TrpValArgTyrAsnGlnPheArgArgGluLeuThrLeuThrValLeu    151015    AspIleValAlaLeuPheSerAsnTyrAspSerArgArgTyrProGly    202530    GlyIleArgThrValSerGlnLeuThrArgGluIleTyrThrAsnPro    354045    ValLeuCysGluAsnPheSerGluAspGlySerPheArgGlyMetAla    505560    GlnArgIleGluGlnAsnIleArgGlnProHisLeuMetAspIleLeu    65707580    AsnSerIleThrIleTyrThrAspValHisArgGlyPheAsnTyrTrp    859095    SerGlyHisGlnIleThrAlaSerProValGlyPheSerGlyProGlu    100105110    PheAlaPheProLeuPheGlyAsnAlaGlyAsnAlaAlaProProVal    115120125    LeuValSerLeuThrGlyLeuGlyIlePheArgThrLeuSerSerPro    130135140    LeuTyrArgTyrThrGlnArgIleIleLeuGlySerGlyProAsnAsn    145150155160    GlnGluLeuPheValLeuAspGlyThrGluAsnAsnPheSerPheAla    165170175    SerLeuThrThrAsnLeuProSerThrIleTyrArgGlnArgGlyThr    180185190    ValAspSerLeuAspValIleProProGlnAspAsnSerValProPro    195200205    ArgAlaGlyLysArgValGluPheSerLeuHisArgLeuSerHisVal    210215220    ThrMetLeuSerGlnAlaAlaGlyAlaValTyrThrLeuArgAlaPro    225230235240    ThrPheSerTrpGlnHisArgSerAlaGluPheAsnAsnIleIlePro    245250255    SerSerGlnSerLeuIleThrGlnIleProLeuThrLysSerThrAsn    260265270    LeuGlySerGlyThrSerValValLysGlyProGlyPheThrGlyGly    275280285    AspIleLeuArgArgThrSerProGlyGlnIleSerThrLeuArgVal    290295300    AsnIleThrAlaProLeuSerGlnArgTyrArgValArgIleArgTyr    305310315320    AlaSerThrThrAsnLeuGlnPheHisThrSerIleAspGlyArgPro    325330335    IleAsnGlnGlyAsnPheSerAlaThrMetSerSerGlySerAsnLeu    340345350    GlnSerGlySerPheArg    355    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 980 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    ATTAAACACTAAATACATTCACATTATTCTAACAAAGAAAAGGAGTAATAATTATGAAAC60    ATCATAAAAATTTTGATCACATAGTTTGGGACTTCGCTGAAAAGTGGACTGAACAAAAGG120    GGGTAGATTTAAAAAGGGTCAGTTATGTAGATCCCATTACTGGTGAAGATACATTAGAGT180    TTATAACCAAATTTAATTATGTTGGGAAATTAGAAGAAAAAGCTTATTGTCCAGAAGTAA240    TAGAAACACAATCTTTTTCAAACTCAAATTGTGACGTTTCGAGGGAATTTCTAAAGAAAA300    AAGTAGACAGGAAGGAATGTTATTTATGGGATATAGACTATGGGTTTATTATACCAACTT360    CGGTACTTACAAATCCATTATTACCCCCCACTCTCAATGAAAAAATTAATCCAGCAATGG420    AAGTGGACTTATTTAAAAGTGCAAACCTGTTTGAATCCAAACTAAATAATTATAGAATGA480    TAGAAGCAGGTGTTTATATTGAACCAAATCAAGCAGTAACCGCCAGCATAATGGTTACAC540    CAAAACAAGTACAGCAAGATTATTGTATTAGCCTTGAGATTTCAGGTAGTATTATCATTG600    AGCTGAAAGATGCTTATAATGCTTGTACAGATAAAGAAACTATTGAAACAATATTCTATA660    CCGTGCCAATTGCAGATATATACAGATCCGAGCTTGCCCATAACCATTCCTTTCATTTAG720    ATGGAGAAACTGTAATATTTACAGGGAAAGGTACGTTTAAAGGCTTAATATGTTCTAATA780    TATTTGTTGAAGGGGAAAGATTCGATTCTCAAACGGGGGAATGTTTGGGGAAATATGTGA840    TCCCATTAAGTATAGAAAAGAAAAATAATGTAGATTGTATCTCTATATTTTTAAATTCAG900    AAAAAGGTGGGATTTAACATGATAGTAGATTTATATAGATATTTAGGTGGATTGGCAGCA960    GTAAATGCCGTACTTCACTT980    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1121 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    GATTTAAACATGATAGTAGATTTATATAGATATTTAGGTGGATTGGCAGCAGTAAATGCC60    GTACTTCACTTTTATGAGCCACGCCCTGATATATGTAGGAATATAAGCGAAGAATATAAC120    CTTATAGTATTTGGAGACCGTATACCAACTTTTAGCATAGATCCTTCGCAAATAAATATT180    AACAATTTATCTGTGGACACTCCAGTGGATGAAATAACTATTAATAACGTGAGAAGTATA240    CAATTAATATCTAGTCGTTTTGAAAATACAGGATTTGTCGATACTGAAAATTATTTTACT300    CCTGAATTATCTAGAACAGTTGTAAATAGCATATCTACATCGACTACTACAGGATATAAG360    TACACTCAATCCCTTACTGTTTCATCCAAATTCTCCTTTAATTTCCCAGTTGCGGGTGCA420    GAAAATAATATTTCATTTTCAGTAGGTTTTGAACAAAACCTTTCAACTACAGAAACTAAA480    ACAGAAAGTACTTCAACGCTTATGCGTATACCTCCACAACCAGTTTCCGTAAGACCCAGA540    ACAGCAAAAAGGGTTGAAATATCGCTCTTTGAATTGGCAATCCCTAGAATACAAAACGAA600    ATTTCCGGATTTGTAACAGGTACTCTTCCAACAATTTCAAATTCGCATATTTCCGATCTT660    TATGCTGTATTAACACGGACTGATAGCCTATGCCCTAATTCATATATTAACCGAGATGAC720    TTTTTAAGAATAGATCATGAAAATAGGGGTTTGGGATTACAAGGCTTCGGTTCTCTCACT780    GGAAATTTAACATCATTAGATTTTGCAATTAGAACTACTGAATATGATTTACCTTCAAAT840    ACAATTATAAATATAGAGAACGAAATAAAAAGAGCCCATATACTCACACAGTAATTAATA900    GAAATAGACCGATAATCGGTCTTCCCCCTGTCAAGTAGGCCTAGTGACAGGGTTCTTGCT960    GTGGACCGCAAGGTAGCAAATTTCTGAAGACCCATATGGGGTACCGTCAGGAAAATGCGG1020    ATTTACAACGCTAAGCCCATTTTCCTGACGATTCCCCCATTTTTAACAACGTTAAGAAAG1080    TTTCAATGGTCTTAAAGAATCTAATGAGATCATTTTCTCCG1121    __________________________________________________________________________

What is claimed is:
 1. A biologically pure Bacillus thuringiensis strainor spores, crystals or mutants thereof having insecticidal activityagainst an insect pest of the order Lepidoptera and an insect pest ofthe order Coleoptera, in which the Bacillus thuringiensis strain isBacillus thuringiensis EMCC0075 having the identifying characteristicsof NRRL B-21019.
 2. A biologically pure Bacillus thuringiensis strain orspores, crystals or mutants thereof having insecticidal activity againstan insect pest of the order Lepidoptera and an insect pest of the orderColeoptera, in which the Bacillus thuringiensis strain is Bacillusthuringiensis EMCC0076 having the identifying characteristics of NRRLB-21020.
 3. An isolated delta-endotoxin having a molecular weight ofabout 33,000 daltons and an amino acid sequence as depicted in SEQ IDNO:
 37. 4. The delta-endotoxin of claim 3 in which the delta-endotoxinis obtained from Bacillus thuringiensis EMCC0075 having the identifyingcharacteristics of NRRL B-21019, or a spore or mutant thereof which hassubstantially the same properties as Bacillus thuringiensis EMCC0075 orBacillus thuringiensis EMCC0076 having the identifying characteristicsof NRRL B-21020, or a spore or mutant thereof which has substantiallythe same properties as Bacillus thuringiensis EMCC0076.
 5. An isolateddelta-endotoxin having a molecular weight of about 33,000 daltons and anamino acid sequence as depicted in SEQ ID NO:
 38. 6. The delta-endotoxinof claim 5 in which the delta-endotoxin is obtained from Bacillusthuringiensis EMCC0075 having the identifying characteristics of NRRLB-21019, or a spore or mutant thereof which has substantially the sameproperties as Bacillus thuringiensis EMCC0075 or Bacillus thuringiensisEMCC0076 having the identifying characteristics of NRRL B-21020, or aspore or mutant thereof which has substantially the same properties asBacillus thuringiensis EMCC0076.
 7. An insecticidal compositioncomprising a delta-endotoxin having a molecular weight of about 33,000daltons and an amino acid sequence as depicted in SEQ ID NO: 37 and adelta-endotoxin having a molecular weight of about 33,000 daltons and anamino acid sequence as depicted in SEQ ID NO: 38 in association with aninsecticidal carrier.
 8. The insecticidal composition of claim 7 inwhich the insecticidal composition further comprises spores of abiologically pure Bacillus thuringiensis strain.
 9. The insecticidalcomposition of claim 8 which further comprises at least twodelta-endotoxins having a molecular weight of about 130,000 and activityagainst an insect pest of the order Lepidoptera.
 10. A method forcontrolling an insect pest of the order Lepidoptera or Coleopteracomprising exposing the pest to an insect-controlling effective amountof an insecticidal composition of claim
 7. 11. A method for controllingan insect pest of the order Lepidoptera comprising exposing the pest toan insect-controlling effective amount of an insecticidal composition ofclaim 8.